Project Summary/Abstract Assembly of major histocompatibility complex (MHC) class I molecules occurs within the endoplasmic reticulum (ER) of cells. Newly synthesized MHC class I molecules are recruited into interactions with the transporter associated with antigen processing (TAP), tapasin, ERp57, protein disulfide isomerase, calnexin and calreticulin. This complex of accessory proteins can be considered a molecular machine whose job it is to (i) pump the peptide products of protein degradation into the region of MHC class I assembly (ii) recruit unassembled MHC class I (iii) facilitate MHC class I-peptide assembly and (iv) ensure regulated release of optimally loaded MHC class I. Much remains to be understood about the workings of this intricate molecular machine, which has been the focus of our research for the past eleven years. Based on our previous work with the TAP transporter, we are able to propose a detailed model for how ATP binding and hydrolysis couple to peptide binding and transport. In the proposed studies we will examine effects of TAP substrates on nucleotide binding and exchange by TAP, and the effects of viral inhibitors upon these processes. A model for the peptide-binding site of TAP will also be examined. These investigations will allow for better understanding of how TAP can be manipulated to enhance or suppress immune responses, and will also allow for better predictions of immunodominant cytotoxic T lymphocyte (CTL) epitopes. We have also been able to reconstitute a tapasin-MHC class I interaction using purified proteins. Tapasin preferentially binds peptidedeficient MHC class I molecules, and the tapasin-MHC class I interaction is regulated by both peptides and the MHC class I light chain, [unreadable]2-microglobulin. Based on analyses of the assembly characteristics of various MHC class I allotypes in tapasin-deficient cells, it is our hypothesis that tapasin is essential for peptide loading of MHC class I allotypes that have inherently slow peptide loading kinetics. These possibilities will be investigated in vitro using different assays systems with reconstituted proteins. We have been able to purify soluble tapasin-MHC class I complexes from cells, in combination with other ER factors. This complex will be further investigated to understand the nature of intermolecular interactions, and the functions of associated proteins. A delicate balance of intracellular interactions appear to regulate assembly and surface expression of MHC class I vs. ER retention in an unassembled state, and we will undertake experiments to elucidate these processes. Although all MHC class I molecules appear to follow the same assembly route within the ER, closely related HLA-B allotypes differ dramatically in their rates of assembly and ER exit. In the studies proposed here, we seek to classify high frequency HLA-B alleles as rapid or slow trafficking, and to also examine the functional consequences of rapid or slow trafficking upon antigen presentation and disease progression. It is our hypothesis that rapid and slow trafficking allotypes have distinct and important functions in the CTL response. Together these studies will allow for a better understanding of the different steps of the MHC class I assembly route, and will contribute to the development of more effective strategies to enhance CTL responses in infection and cancer.